CURLY LEAF is required for the auxin-dependent regulation of 3-dimensional growth specification in Physcomitrium patens

The no gametophores 4 ( nog4-R ) mutant cannot make the transition from 2-dimensional (2D) to 3-dimensional (3D) growth in Physcomitrium patens and forms side branch initials that are largely fated to become sporophyte-like structures. We describe the three different developmental trajectories adopted by the nog4-R mutant, all of which result in indeterminate growth and defects in cell division plane orientation. A candidate gene approach confirmed that the causative mutation resided in the CURLY LEAF gene, and we highlight a previously uncharacterized role for CURLY LEAF in maintaining auxin homeostasis in P. patens .


4/17/2023 -Open Access
A) The three developmental trajectories observed in the nog4-R mutant (lanceolate, unicorn and minotaur) at the 2-cell, 3-cell, 5-10 cell and 10+ cell stages and an overview of an intermediate stage of development. A schematic has been included to enable comparisons more clearly with the predictable pattern of development observed in wild type. Developing 'buds' have been stained with propidium iodide and imaged using confocal microscopy. In the 2-cell and 3-cell images, the most recent divisions have been indicated by yellow arrows. A blue dot denotes the rhizoid initial cell. Scale, 20 µm. B) Representative images of one month old nog4-R tissues grown in the presence or absence of the auxin analogue NAA (100 nm). Scale, 50 µm. C) The proportion of side branches that give rise to 'buds' in wild type and nog4-R (no. buds formed per 15 cells), in the presence or absence of 100 nm NAA or 100 nm BAP (a cytokinin analogue). D) Comparison of the PpCLF transcript sequence in wild type and in the nog4-R mutant. Numbers indicate the relative position of the codon in the PpCLF transcript sequence (note the presence of a premature stop codon in nog4-R at position 104).

Description
The colonization of land by plants was a pivotal moment in the history of life on Earth. The transition from aquatic to terrestrial environments was facilitated by the evolution of 3-dimensional (3D) growth; more specifically, the ability to rotate the division plane of an apical cell. Consequently, plants developed complex and diverse morphologies that enabled them to survive more effectively in harsh environments, located away from the water's edge (Graham et al., 2000;Delwiche and Cooper 2015;Whitewoods et al., 2018).
We recently developed the bryophyte Physcomitrium patens as a model system to genetically dissect 3D growth (reviewed elsewhere - Moody, 2019;Moody, 2022). P. patens has a protracted 2-dimensional (2D) filamentous growth phase (the tip growing protonema) that precedes the transition to 3D growth, and this can be continually maintained in tissue culture. The protonema initially comprises chloronemal cells, which emerge from spores, but later differentiates into caulonemal cells. From the latter emerge side branch initials that can form either secondary protonema (95%) or shoots (gametophores) that develop from gametophore initial cells (5%). To establish 3D growth, a gametophore initial cell divides obliquely to form an apical and a basal cell, which then both divide obliquely (perpendicularly to the first division plane) to form a 4-cell bud. The apical cell then undergoes two successive rotating divisions to specify an apical cell with a tetrahedral shape, which can divide to self-renew and generate the cells that comprise the leaf-like phyllids that wrap around the central axis of the gametophore in a spiral phyllotaxy (Harrison et al., 2009;Tang et al., 2020) (Figure 1A). A fine balancing act between auxin and cytokinin ensures that the correct number of gametophores are formed, cell walls are correctly oriented in developing gametophores, and that stemness of the tetrahedral apical cell is maintained (Ashton et al., 1979;Schulz et al., 2000;Thelander et al., 2019;Hyoung et al., 2020). Important regulators of the initiation, establishment, and maintenance of 3D growth include PpAPB1-4 (Aoyama et al., 2012), PpMACRO2 (Wang et al., 2020), the glycerol-3-phosphate acyltransferases PpGPAT2 and PpGPAT4 (Lee et al., 2020), Defective Kernel 1 Demko et al., 2014;Johansen et al., 2016;Perroud et al., 2020), CLAVATA (Whitewoods et al., 2018;Cammarata et al., 2022), the exocyst subunit PpSEC6 (Brejšková et al., 2021), the microtubule-associated protein targeting factor for Xklp2 (TPX2; Kozgunova et al., 2022), and NO GAMETOPHORES 1 (Moody et al., 2018) and NO GAMETOPHORES 2 (also known as PpHCT; Moody et al., 2021;Kreigshauser et al., 2021).
To identify additional regulators of 3D growth, we UV-mutagenized a wild-type strain of P. patens (Gd::GFP; Perroud et al., 2011) and screened for mutants that failed to establish 3D growth. As a result of this screen, we identified the 'no gametophores 4 -Reference' (nog4-R) mutant, which exhibits an array of striking developmental defects. Notably, in nog4-R mutants, side branch initials that normally give rise to filaments or buds are largely displaced by sporophyte-like indeterminate structures that can neither establish 3D growth nor develop gametophores ( Figure 1A). We have observed three distinct developmental trajectories adopted by these sporophyte-like structures in the nog4-R mutant, referred to as the lanceolate, unicorn and minotaur. The lanceolate structure is derived from a small isodiametric gametophore initial cell that divides obliquely as in wild type, but then fails to establish a clearly defined apical and basal cell. Successive divisions predominantly occur perpendicularly to the first two division planes and is accompanied by a marked reduction in cell expansion. This results in the formation of a compact and bulbous structure that resembles the head of a lance, and in some cases, multiple lanceolate structures can form from a single gametophore initial cell. A unicorn is derived from a bulbous gametophore initial cell, but the first division is roughly parallel to the parent caulonemal cell and is not characteristically oblique. Subsequent divisions establish an apical cell that divides to self-renew but then yields cells from only two faces, seemingly in two continuous cell files (2D growth). A minotaur is derived from a gametophore initial cell that appears, at first, to follow the trajectory towards 3D growth; the first division is characteristically oblique, and the second division is perpendicular to the first division. However, the cell that would ordinarily transition into a rhizoid initial cell eventually switches fate to become an extra apical cell. Consequently, a bifurcation event is triggered, and this results in the formation of a two-horned bud with bilateral symmetry; 3D growth is never fully specified or established. We have observed that, new cell walls that form at the earliest stages of the development of either lanceolate, unicorn or minotaur structures are curved and wavy, like those observed in the Δdek1 mutant . Furthermore, both unicorn and minotaur structures follow an indeterminate growth programme, and it is not unusual to detect structures that exceed 700 µm in length ( Figure 1A).
The nog4-R mutant phenotypes observed were highly reminiscent of those mutants with underlying defects in auxin and/or cytokinin signaling. Thus, we explored whether the nog4-R mutant could respond to auxin or cytokinin in an appropriate manner. We discovered that exogenous auxin treatment can strongly suppress the outgrowth of the indeterminate sporophytelike structures to restore filamentous growth to the mutants ( Figure 1B). This implies that the nog4-R mutant is defective in maintaining auxin homeostasis similarly to the recently described nog2-R mutant, which fails to inhibit supernumerary gametophore initial cell formation (Moody et al., 2021). We went on to quantify the effects of auxin and cytokinin treatment on the proportion of side branches that are fated to become a bud; it has previously been shown that approximately one in 15 side branch initials are fated to become a gametophore in wild type (Aoyama et al., 2012;Perroud et al., 2014). We performed an assay to calculate the proportion of side branches that initiate buds versus those that form filaments and compared wild type to the nog4-R mutant in the presence and absence of auxin or cytokinin. In wild type, we observed that the proportion of side branches that form buds is mildly reduced in response to auxin treatment but is strongly induced by cytokinin (approximately 60% of the filaments formed at least one bud and some formed as many as four buds). Conversely, in nog4-R, the proportion of side branches that form buds is extremely high in the absence of either auxin or cytokinin (approximately 95% filaments formed at least one bud and some formed as many as six buds), is strongly reduced in response to auxin treatment (approximately 60% filaments formed at least one bud and some formed as many as three buds) and is somewhat unaffected by cytokinin treatment ( Figure 1C). These data suggest that endogenous levels of cytokinin within the nog4-R mutants may be disproportionately high, and that auxin treatment is required to readdress the balance between auxin and cytokinin.
It is known that the polycomb repressive complex 2 (PRC2) components FERTILIZATION INDEPENDENT ENDOSPERM (PpFIE) and CURLY LEAF (PpCLF) are required to suppress the formation of sporophyte-like apical cells within the gametophyte generation of P. patens (Mosquna et al., 2009;Okano et al., 2009;Pereman et al., 2016). PpFIE and PpCLF are represented by single copy genes in P. patens, and disruption of either of these genes results in the formation of sporophytelike apical cells in the gametophyte generation. In the gametophyte, these apical cells exhibit uncontrolled indeterminate growth. This is quite unlike a true sporophyte apical cell, which undergoes a brief determinate growth programme before a seta meristem is formed and subsequently a mature sporangium (Sakakibara et al., 2008). We realised that the nog4-R mutant bore a striking resemblance to those mutants lacking a functional copy of either the PpCLF or PpFIE genes. Thus, we took a candidate gene approach to determine whether nog4-R contained UV-induced mutations within the coding sequences of either PpCLF or PpFIE. We discovered that the coding sequence of PpFIE aligned perfectly with the wild-type sequence and therefore did not contain any UV-induced mutations. However, a G>A transition generated a nonsense mutation (W 104 Ter) in the coding sequence of the PpCLF gene ( Figure 1D). Thus, we have demonstrated that a mutated PpCLF gene was responsible for the nog4-R mutant phenotype.
In summary, loss of PpCLF function leads to the excessive formation of gametophore initial cells, and cell division orientation defects prevent the specification of a tetrahedral-shaped apical cell that is required for 3D growth. Furthermore, supernumerary gametophore initial cell formation can be suppressed by exogenous auxin treatment. Thus, PpCLF is required to specify 3D growth in an auxin-dependent manner.

UV mutagenesis
A 1% Driselase solution (Sigma-Aldrich, Cat. No. D9515) was prepared in 8% mannitol and incubated at room temperature for 15 min before centrifugation at 3,300 xg for 3 min. The supernatant was filter sterilized through a 0.22 µm syringe filter into a sterile tube. To isolate protoplasts, the Gd::GFP marker line (Perroud et al., 2011), grown for one week on cellophane overlaid BCDAT, was added to the Driselase solution, wrapped in foil and then gently agitated for 30-40 min at room temperature. The protoplast suspension was passed through a 100 µm cell strainer, pelleted by centrifugation at 120 xg for 3 min, and then washed twice with 8% mannitol (repeating the centrifugation in between each wash step). The cells were counted using a haemocytometer, and the cell density adjusted to 5x10 4 cells mL -1 . 1 mL of the cell suspension was then added to individual petri dishes containing cellophane overlaid PRMB medium (BCDAT supplemented with 10 mM CaCl 2 , 6% mannitol and 0.5% glucose). These plates were then exposed to a 75000 µJ dose of UV radiation using a Stratalinker (lids off). Irradiated plates were incubated at 24 o C in the dark for 24-48 h and then transferred to the light for a further 2-3 weeks to allow protoplast regeneration. Individual regenerating protoplasts were transferred to wells of a 24 well plate containing BCD and grown under standard conditions for 4-6 weeks, and then phenotypically characterized. Those mutants that were unable to produce gametophores were selected for long term growth on BCD medium.

Microscopy
To check cell wall division orientation, tissues were grown for two weeks on BCD medium and then stained using 10 μgmL -1 propidium iodide (PI) for 2 minutes. Tissues were then mounted in water and imaged using a Leica SP5 confocal microscope. PI was excited at 488 nm with 20% laser power, and a 63x water immersive lens was used in detecting PI at 600-630 nm. All other images were captured using either a Leica DMRB microscope or a Leica M165C stereomicroscope, equipped with a QImaging Micropublishing 5.0 RTV camera.

Bud assays
To quantify the proportion of side branches that give rise to filaments, tissues (wild type and nog4-R) were homogenized and plated onto BCD plates supplemented with 100 nM 6-benzylaminopurine (BAP; synthetic cytokinin), 100 nM 1naphthaleneacetic acid (NAA; synthetic auxin) or a minimal amount of 70% ethanol (solvent control). Three-week-old tissues were mounted on a slide in water to determine budding frequency. This was performed by determining the buds produced on side branches within a stretch of 15 caulonemal cells. Four biological replicates (colonies) were performed, and 10 filaments identified each time (n=40).

Cloning of PpFIE and PpCLF
RNA was extracted from one week old wild-type and nog4-R tissues grown on cellophane overlaid BCDAT, using the RNeasy Kit (Qiagen). RNA was DNase treated using TURBO DNase (Ambion) prior to cDNA synthesis using Superscript III Reverse Transcriptase (Invitrogen). Full length PpFIE and PpCLF transcripts were PCR amplified from cDNA using Verifi TM PCR Mix (PCRBIO) according to the manufacturer's instructions. PpFIE was amplified using FIE_1.1K_FOR (ATGGCCCTGAAGCCTGC) and FIE_1.1K_REV (TCAAGACACAGCATCCCAGC), and PpCLF was amplified using CLF_3K_FOR (ATGGCGTCCTCCAGCTACG) and CLF_3K_REV (TTAAGCAACTTTCTGTGCTCGTC). PCR products were sequenced directly by Sanger sequencing (Source Bioscience).